Research ArticleCancer

Eradication of spontaneous malignancy by local immunotherapy

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Science Translational Medicine  31 Jan 2018:
Vol. 10, Issue 426, eaan4488
DOI: 10.1126/scitranslmed.aan4488
  • Fig. 1 CpG induces the expression of OX40 on CD4 T cells.

    (A) A20 tumor–bearing mice were treated either with vehicle (top) or CpG (middle). Forty-eight hours later, tumors were excised and a single-cell suspension was stained and analyzed by flow cytometry. (B) OX40 expression within the CD3+CD4+ subset was separately analyzed for FoxP3-negative [effector T cell (Teff)] and FoxP3-positive [regulatory T cell (Treg)] subsets. Fold changes of OX40+ cells were calculated according to their frequencies in the vehicle versus CpG treatment (n = 2). (C) Fine needle aspirates from CpG-injected and noninjected tumors of a follicular lymphoma patient were obtained 22 hours after treatment. Fluorescence-activated cell sorting (FACS) plots of OX40 expression within the CD4+ subset after a 24-hour rest in media. Top: Nontreated lesion. Bottom: CpG-treated site (n = 2). (D) Single-cell suspensions from biopsy specimens of human lymphoma (five mantle cell lymphomas and five follicular lymphomas) were exposed in vitro to CpG for 48 hours and analyzed for OX40 expression as in (B). (E) CpG-stimulated human lymphoma–infiltrating CD4+ T cells, CD8+ T cells, and CD19+ B cells were gated and visualized in tSNE (t-Distributed Stochastic Neighbor Embedding) space using Cytobank software. The viSNE map shows the location of each CD4+, CD19+, and CD8+ cell population (green, blue, and orange, respectively; bottom). Cells in the viSNE maps were colored according to the intensity of OX40 expression. CpG up-regulation of OX40 expression on a subset of CD4+ T cells is highlighted by a red box. (F) BALB/c mice were implanted subcutaneously with A20 lymphoma cells (5 × 106) on both the right and left shoulders. When tumors reached between 0.7 and 1 cm in the largest diameter (typically on days 8 to 9 after inoculation), phosphate-buffered saline and CpG (50 μg) were injected into one tumor site (left tumor). Sixteen hours later, 64Cu-DOTA-OX40 was administered intravenously via the tail vein. Positron emission tomography imaging of mice was performed 40 hours after in situ treatment. Left: Vehicle-treated. Right: CpG-treated. These images are representative of six mice per group. (G) Fresh A20 tumors were excised from animals (typically 5 to 6 days after inoculation), and either whole tumors (left), T cells purified from the tumor (middle), or whole tumor depleted of CD11b- and CD11c-expressing cells (right) were treated for 48 hours with media (top) or CpG (bottom) and were analyzed for their expression of OX40 by flow cytometry. (H) Left: A20 tumors were excised as in (F). Right: Single-cell suspensions from biopsy specimens of human follicular lymphoma. Tumors were treated for 48 hours with media and CpG with or without antibodies (1 μg/ml) to interleukin-2 (IL-2), IL-4, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, interferon-γ (IFN-γ), or tumor necrosis factor–α (TNF-α) and were analyzed for their expression of OX40 by flow cytometry. α–IL-12, *P = 0.0144; α–IFN-γ, **P = 0.0032; α–TNF-α, **P = 0.008, unpaired t test, either depleting antibody versus CpG alone.

  • Fig. 2 In situ vaccination of CpG in combination with anti-OX40 antibody cures established local and distant tumors.

    (A) Treatment schema. BALB/c mice were implanted subcutaneously with A20 lymphoma cells (5 × 106) on both the right and left sides of the abdomen. When tumors reached between 0.5 and 0.7 cm in the largest diameter (typically on days 4 to 5 after inoculation), αOX40 (4 μg) and CpG (50 μg) were injected into one tumor site every other day for a total of three doses. Tumors sizes were serially measured with a caliper. (B) Tumor growth curves. Left column: Treated tumors (Tr). Right column: Nontreated tumors (NT). Top to bottom: Vehicle, CpG, αOX40, and CpG and αOX40 and survival plots of the treated mice (n = 10 mice per group). ****P < 0.0001, unpaired t test. Shown is one representative experiment out of nine. (C) Effect of CD4/CD8 depletion. Mice were implanted with bilateral tumors, and one tumor was injected with CpG and αOX40 antibody according to the schema in (A). CD4 (0.5 mg)–and CD8 (0.1 mg)–depleting antibodies were injected intraperitoneally on days 6, 8, 12, and 15 (n = 10 mice per group). (D) CD8 T cell immune response. Splenocytes from the indicated groups obtained on day 7 after treatment were cocultured with media, 1 × 106 irradiated 4T1 cells (unrelated control tumor), or A20 cells (homologous tumor) for 24 hours. Intracellular IFN-γ was measured in CD8+ T cells by flow cytometry as a percentage of CD44hi (memory CD8) T cells shown in dot plots and bar graph, summarizing data from three experiments (n = 9 mice per group). ****P < 0.0001, unpaired t test.

  • Fig. 3 In situ vaccination with CpG and anti-OX40 is therapeutic in a spontaneous tumor model.

    (A) MMTV-PyMT transgenic female mice were injected into the first arising tumor (black arrow) with either vehicle (top) or with CpG and αOX40 (bottom); pictures were taken on day 80. (B) CpG and αOX40 decrease the tumor size of a nontreated contralateral tumor. Growth curves represent the volume of a contralateral (untreated) tumor in mice that had two palpable tumors at the beginning of treatment. Mice treated by in situ vaccination (red; n = 6) or vehicle (black; n = 6). ***P = 0.0008, unpaired t test. (C) CpG and αOX40 decrease the total tumor load. Growth curves represent the sum of the volume of 10 tumors from the different fat pads of each mouse, measured with calipers (n = 10 mice per group), and the window of treatment is indicated by the gray bar. ****P < 0.0001, unpaired t test. (D) Time-matched quantification of the number of tumor-positive mammary fat pads. **P = 0.011, unpaired t test (n = 9 mice per group). (E) Mice were sacrificed at the age of 80 days, and lungs were excised and analyzed ex vivo for the number of metastases (mets). ****P < 0.0001, unpaired t test (n = 10 mice from vehicle-treated group; n = 9 mice with CpG and αOX40). (F) Survival plots of the treated mice. ****P < 0.0001. Data are means ± SEM (n = 10 mice per group). (G) CD8 T cell immune response. Splenocytes from the indicated groups obtained on days 7 to 15 after treatment were cocultured for 24 hours with either media or 1 × 106 irradiated tumor cells taken from an independent contralateral site on the body. Intracellular IFN-γ was measured in CD8+ T cells by flow cytometry as shown in dot plots and bar graph, summarizing data as a percentage of CD44hi (memory CD8) T cells (n = 3 mice per group).

  • Fig. 4 Immunizing effects of intratumoral CpG and anti-OX40 are local and tumor-specific.

    (A) Three-tumor model. Each mouse was challenged with three tumors, two of them A20 lymphoma (blue) and one CT26 colon cancer (red). Mice were treated at the indicated times (black arrows). Tumor growth curves of the treated tumor (bottom left), the homologous nontreated A20 tumor (top right), and the heterologous CT26 tumor (bottom right). Photos of a representative mouse at day 11 after tumor challenge from the vehicle-treated group and from the group with A20 tumors treated with intratumoral CpG and αOX40 (n = 10 mice per group) are shown. (B) Reciprocal three-tumor model with two CT26 tumors and one A20 tumor. Treatment was given to one CT26 tumor, and growth curves are shown for the treated CT26 tumor site (bottom right), the nontreated homologous CT26 tumor site (top right), and the heterologous A20 tumor (bottom right). Photos of a representative mouse from this experiment (n = 10 mice per group) are shown. (C) Mixed three-tumor model. Each mouse was challenged with three tumors: one A20 (blue, top right abdomen), one CT26 (red, bottom right abdomen), and one mixture of A20 and CT26 tumor cells (blue and red gradient, left abdomen). Mice were treated only in the mixed tumor at the indicated times (black arrows). Tumor growth curves of the treated tumor (bottom left), the nontreated A20 tumor (top right), and the nontreated CT26 tumor (bottom right). Photos of a representative mouse at day 11 after tumor challenge from the vehicle-treated group (top) and at day 17 from the intratumoral CpG and αOX40 (n = 8 mice per group) are shown.

  • Fig. 5 A competent Fc is required for the antitumor immune response.

    (A and B) Effect of Treg depletion. (A) Tumors were implanted according to the schema in Fig. 2A. Mice were treated with either CpG and anti–folate receptor 4 (FR4) antibody (15 μg) or CpG and αOX40 as described in Fig. 2A, and the NT was measured over time. ****P < 0.0001, unpaired t test (n = 10 mice per group). (B) DEREG mice were implanted with B16-F10 melanoma cells (0.05 × 106) on both the right and left sides of the abdomen. Diphtheria toxin (DT; 1 μg) was injected intraperitoneally on days 1, 2, 7, and 14. CpG or combination of CpG and anti-OX40 was given on days 7, 9, and 11. The NT was measured over time. *P = 0.0495, unpaired t test (n = 4 mice per group). (C) A20 cells were inoculated and treated as described in Fig. 2A, tumor volumes were measured after treatment of CpG with either αOX40 rat immunoglobulin G1 (IgG1) (red) or αOX40 rat IgG1 Fc mutant (black). ****P < 0.0001, unpaired t test (n = 10 mice per group). WT, wild type. (D) Tumors from control and treated mice were excised at the indicated times after a single treatment, and the cell populations from the different groups were differentially labeled (barcoded) with two different levels of violet tracking dye (VTD) and mixed together, stained, and analyzed as a single sample [n = 3 mice per group (C to F)]. (E to H) Dot plots for single time point and bar graphs for replicates of multiple time points. (E) Number of F4/80 CD11b+ myeloid cells. **P = 0.009 (8 h), Fc WT versus vehicle. (F) CD137 expression on natural killer (NK) cells. **P = 0.0035 (2 h), *P = 0.0343 (8 h), unpaired t test, Fc WT versus Fc mutant. (G) CD69 expression on CD8+ T cells. *P = 0.025 (8 h), **P = 0.0064 (24 h), unpaired t test, Fc WT versus Fc mutant. (H) Treg cell proliferation. ***P = 0.0003 (24 h), unpaired t test, Fc WT versus Fc mutant.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/426/eaan4488/DC1

    Fig. S1. In situ vaccination with a TLR9 ligand induces the local expression of OX40 but not that of PD1 or CTLA4.

    Fig. S2. CpG induces the expression of OX40 on CD4 T cells.

    Fig. S3. Cytokines are playing a role in the CpG T cell cross-talk.

    Fig. S4. Intracellular IFN-γ production of CD4+ cells.

    Fig. S5. Frequency of T cell subsets.

    Fig. S6. Tumor recurrence is sensitive to treatment with anti-OX40 and CpG.

    Fig. S7. Resiquimod (R848) in combination with anti-OX40 and anti-PD1/PDL1 in combination with CpG.

    Fig. S8. In situ vaccination with CpG and anti-OX40 is effective against breast carcinoma, colon cancer, and melanoma.

    Fig. S9. Dose de-escalation of CpG and αOX40 antibody.

    Fig. S10. Systemic administration of anti-αOX40 antibody.

    Fig. S11. Long-term memory in cured mice.

    Fig. S12. Confirmation of Treg depletion from the tumor.

    Fig. S13. Anti-OX40 antibody stimulates Teffs and inhibits function of Tregs.

    Fig. S14. Requirement for Fc competency of the anti-OX40 antibody.

    Fig. S15. Neither Tregs nor Teffs are depleted by anti-OX40 antibody.

    Table S1. Primary data.

  • Supplementary Material for:

    Eradication of spontaneous malignancy by local immunotherapy

    Idit Sagiv-Barfi, Debra K. Czerwinski, Shoshana Levy, Israt S. Alam, Aaron T. Mayer, Sanjiv S. Gambhir, Ronald Levy*

    *Corresponding author. Email: levy{at}stanford.edu

    Published 31 January 2018, Sci. Transl. Med. 10, eaan4488 (2018)
    DOI: 10.1126/scitranslmed.aan4488

    This PDF file includes:

    • Fig. S1. In situ vaccination with a TLR9 ligand induces the local expression of OX40 but not that of PD1 or CTLA4.
    • Fig. S2. CpG induces the expression of OX40 on CD4 T cells.
    • Fig. S3. Cytokines are playing a role in the CpG T cell cross-talk.
    • Fig. S4. Intracellular IFN-γ production of CD4+ cells.
    • Fig. S5. Frequency of T cell subsets.
    • Fig. S6. Tumor recurrence is sensitive to treatment with anti-OX40 and CpG.
    • Fig. S7. Resiquimod (R848) in combination with anti-OX40 and anti-PD1/PDL1 in combination with CpG.
    • Fig. S8. In situ vaccination with CpG and anti-OX40 is effective against breast carcinoma, colon cancer, and melanoma.
    • Fig. S9. Dose de-escalation of CpG and αOX40 antibody.
    • Fig. S10. Systemic administration of anti-αOX40 antibody.
    • Fig. S11. Long-term memory in cured mice.
    • Fig. S12. Confirmation of Treg depletion from the tumor.
    • Fig. S13. Anti-OX40 antibody stimulates Teffs and inhibits function of Tregs.
    • Fig. S14. Requirement for Fc competency of the anti-OX40 antibody.
    • Fig. S15. Neither Tregs nor Teffs are depleted by anti-OX40 antibody.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Primary data (provided as an Excel file)..

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